Bile acids such as taurocholic acid are organic anions that play a critical role in numerous physiological processes such as a) digestion, b) formation of bile, which functions as an secretory vehicle for numerous compounds such as cholesterol and metabolites of exogenous drugs and carcinogens, and c) regulation of cholesterol homeostasis by modulating its synthesis and degradation. Defects in the transport of bile acids result in intrahepatic cholestasis and numerous pathological conditions including liver injury and failure. A sinusoidal plasma membrane protein mediating sodium-dependent taurocholate uptake by hepatocytes has been isolated and shown to be indistinguishable from microsomal epoxide hydrolase (mEH). This protein, which also mediates the transport of taurocholate into vesicles derived from the smooth endoplasmic reticulum has been shown to exist in two distinct topological orientations in this cell compartment, one of which is targeted to the plasma membrane. The long term goals of this project are to investigate the molecular mechanism and regulation of this important liver transport system as well as delineating its physiological role in the uptake of bile acids and related substrates by a) characterizing the architecture, substrate binding site and functional properties of this transport system in the plasma membrane and endoplasmic reticulum; b) defining the mechanism which regulates the generation of multiple topologies and targeting of this single protein and c) elucidating the mechanism of transcellular movement of bile acids from the blood to the bile compartment. The specific aims of this proposal are: 1) to characterize architectural and functional aspects of this bile acid transport (BAT) protein as expressed in hepatocyte sinusoidal plasma membranes and smooth endoplasmic reticulum membranes using a) sequence- specific anti-peptide antibodies (SSAPA) in conjunction with confocal fluorescence microscopy and immunogold electron microscopy, and b) site- specific and membrane domain-specific chemical and enzymatic reagents; 2) to characterize the topology, transport function and substrate specificity of the BAT protein expressed in the basolateral plasma membrane and endoplasmic reticulum of MDCK cells, and the role of intracellular vesicles in the transcellular movement of bile acids, using cDNA transfection procedures in conjunction with SSAPA and confocal microscopy and 3) to evaluate the role of specific amino acid residues in a) determining the membrane topology and cell targeting of BAT and b) the mechanism of a bile acid transport, using site-directed mutagenesis and transfection procedures. These studies will provide a detailed understanding of the structure, mechanism of action and physiological role of this important transport system, as well as elucidating factors involved in regulating transport characteristics, membrane protein topological orientation and targeting.